Selective cell therapy for the treatment of renal failure

ABSTRACT

Provided herein are isolated populations of kidney cells harvested from differentiated cells of the kidney, wherein cells have been expanded in vitro. The kidney cells may include peritubular interstitial cells of the kidney, and preferably produce erythropoietin (EPO). The kidney cells may also be selected based upon EPO production. Methods of producing an isolated population of EPO producing cells are also provided, and methods of treating a kidney disease resulting in decreased EPO production in a patient in need thereof are provided, including administering the population to the patient, whereby the cells produce EPO in vivo.

RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 12/134,813, filed Jun. 6, 2008, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 60/942,716,filed Jun. 8, 2007, the disclosure of each of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is in the field of selective cell therapy for therestoration of organ function.

BACKGROUND OF THE INVENTION

Chronic renal failure is characterized by a gradual loss in kidneyfunction, and may eventually progress to end stage renal failure, wherethe kidney no longer functions at a level to sustain the body. End stagerenal failure is a devastating disease that involves multiple organs inaffected individuals. The most common cause of end stage renal diseasein the U.S. is diabetes.

One of the functions performed by the kidney is the production oferythropoietin (EPO). When the kidney is functioning properly, lowtissue oxygenation in the renal interstitium stimulates the interstitialcells to produce EPO. The secreted EPO in turn stimulates red blood cellproduction in the bone marrow, which restores tissue oxygen tension tonormal levels. Anemia caused by ineffective hematopoiesis is one of theinevitable outcomes of chronic renal failure due to the kidney'sdecreased ability to produce EPO. EPO has also been reported to protectagainst oxidative stress and apoptosis.

The kidney is the primary producer of EPO in the body and is therefore aprimary target of treatment for renal failure induced anemia. Althoughdialysis can prolong survival for many patients with end stage renaldisease, only renal transplantation can currently restore normalfunction. However, renal transplantation is severely limited by acritical donor shortage.

Treatments used to alleviate anemia associated with renal failure overthe years include repeated transfusions of red blood cells andadministration of testosterone and other anabolic steroids. However,none of these modalities has been entirely satisfactory. Patientsreceiving repeated transfusions are subject to iron overload, and maydevelop antibodies to major histocompatibility antigens. Testosteronehas a minimal effect on erythropoeisis in the bone marrow, and it isassociated with undesirable, virilizing side effects.

Previous efforts to mitigate anemia associated with renal failure haveincluded the administration of purified recombinant EPO (See, e.g., U.S.Pat. No. 6,747,002 to Cheung et al., U.S. Pat. No. 6,784,154 toWestenfelder). However, the administration of recombinant EPO onlyelevates EPO levels in the blood temporarily, and may lead to irondeficiency. Gene therapy approaches have also been pursued, in which EPOis produced using transfected host cells (See, e.g., U.S. Pat. No.5,994,127 to Selden et al., U.S. Pat. No. 5,952,226 to Aebischer et al.,U.S. Pat. No. 6,777,205 to Carcagno et al.; Rinsch et al. (2002) KidneyInternational 62:1395-1401). However, these approaches involve thetransfection of non-kidney cells, and require techniques such as cellencapsulation to prevent antigen recognition and immune rejection upontransplantation. Also, transfection with exogenous DNA may be unstable,and the cells may lose their ability to express EPO over time.

Renal cell-based approaches to the replacement of kidney tissue islimited by the need to identify and expand renal cells in sufficientquantities. In addition, the culturing of renal cells for the purpose ofkidney tissue engineering is particularly difficult, owing to thekidney's unique structural and cellular heterogeneity. The kidney is acomplex organ with multiple functions, including waste excretion, bodyhomeostasis, electrolyte balance, solute transport, as well as hormoneproduction.

There remains a great need for alternative treatment options toalleviate anemia caused by the failure of renal cells to producesufficient amounts of erythropoietin.

SUMMARY OF THE INVENTION

Provided herein in embodiments of the present invention are isolatedpopulations of kidney cells harvested from differentiated cells of thekidney that have been passaged and/or expanded in vitro. In someembodiments, the kidney cells include peritubular interstitial and/orendothelial cells of the kidney. In some embodiments, the kidney cellsconsist of or consist essentially of peritubular interstitial and/orendothelial cells of the kidney harvested from kidney tissue andpassaged in vitro. In some embodiments, cells produce erythropoietin(EPO). In further embodiments, kidney cells are selected for EPOproduction.

Also provided are methods of producing an isolated population of EPOproducing cells, including the steps of: 1) harvesting differentiatedkidney cells; and 2) passaging the differentiated kidney cells, whereinthe cells produce EPO after said passaging; thereby producing anisolated population of EPO producing cells. In some embodiments themethods further include the step of selecting the differentiated kidneycells for EPO production. In some embodiments, the passaging stepincludes growth of differentiated kidney cells in a medium comprisinginsulin transferrin selenium (ITS).

Methods of treating a kidney disease or other ailment, which disease orailment results in decreased EPO production in a subject (e.g., apatient) in need thereof are also provided, including the steps of: 1)providing an isolated population of EPO producing cells; and 2)administering the population to the subject (e.g., in an amounteffective to treat the kidney disease and/or the decreased EPOproduction), whereby the EPO producing cells produce EPO in vivo. Insome embodiments, the providing step is performed by harvestingdifferentiated kidney cells of the kidney and passaging the cells invitro. In some embodiments, the population of EPO producing cellsincludes, consists of or consists essentially of differentiatedperitubular endothelial and/or interstitial cells harvested fromdifferentiated cells of the kidney and passaged in vitro. In someembodiments, the population is provided in a suitable carrier (e.g., acollagen gel) for administration. In some embodiments, the administeringstep is carried out by implanting the population of cells into thekidney of the patient. In some embodiments, the administering step iscarried out by subcutaneously injecting or implanting said composition.In some embodiments, the EPO producing cells are human.

Further provided are isolated populations of cells includingdifferentiated human kidney cells harvested from human kidney tissue andpassaged in vitro. In some embodiments, the kidney cells consist of orconsist essentially of peritubular interstitial and/or endothelial cellsof the kidney harvested from kidney tissue and passaged in vitro. Insome embodiments, the differentiated human kidney cells produceerythropoietin (EPO). In some embodiments, the human kidney cells havebeen passaged from 1-20 times. In some embodiments, the human kidneycells have been passaged at least 3 times. In some embodiments, thepopulation has been selected for EPO production. Some embodiments aresubject to the proviso that the cells are not transfected with anexogenous DNA encoding a polypeptide.

Compositions comprising the population of human kidney cells asdescribed herein and a pharmaceutically acceptable carrier are alsoprovided. In some embodiments, the carrier comprises collagen.

Another aspect of the present invention is the use of the methods asdescribed herein for the preparation of a composition or medicament foruse in treatment or for carrying out a method of treatment as describedherein (e.g., for treating a kidney disease or other ailment resultingin decreased EPO production), or for making an article of manufacture asdescribed herein.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1. Mechanism of erythropoietin (EPO) production. Renal interstitialperitubular cells of the kidney detect low blood oxygen levels, and EPOis secreted into the blood. EPO stimulates the proliferation anddifferentiation of erythroid progenitors into reticulocytes, andprevents apoptosis, causing more reticulocytes to enter the circulatingblood. The reticulocytes differentiate into erythrocytes, increasing theerythron size. Oxygen delivery to the tissues is thereby increased.

FIG. 2. Intracellular erythropoietin immunoreactivity was confirmed inthe primary culture of renal cells at passage 1 (P1), passage 2 (P2) andpassage 3 (P3), compared to the negative control (X400).

FIG. 3. Microscopy images of erythropoietin expressing cells in kidneytissue (left panel) and in cultured kidney cells (right panel).

FIG. 4. Quantification of erythropoietin (EPO) producing cells. Thenumber of cells expressing EPO decreased with the subsequent passages (*p<0.05).

FIG. 5. Western blot analysis of detergent-solubilized cell extractsdetected EPO protein (34 kDa) of early passage primary cultured renalcells (P0-P3).

FIG. 6. EPO expression analysis using FACS. Top Row: Mouse cells,passages 0-3. Bottom Row: Rat cells, passages 0-3.

FIG. 7A-7B. Mouse renal cell characterization. EPO expression isconfirmed by immunofluorescence (FIG. 7A) (KNRK cells were used aspositive control). GLEPP1 and Tamm Horsfall kidney markers were alsodetected (FIG. 7B).

FIG. 8. Rat renal cell characterization. Cultured rat kidney cells havevarious cell morphologies shown by phase contrast microscope (leftpanels), and express GLEPP1 and Tamm Horsfall kidney markers (rightpanels).

FIG. 9. EPO expression in HepG2 cells was shown by western blot andcompared with EPO expression in kidney tissue.

FIG. 10. EPO protein expression of cultured cells under hypoxicconditions. Lewis rat kidney cells and HepG2 cells were cultured undernormal and hypoxic conditions, and EPO production was assessed bywestern blot of cells. 34 kDa=EPO; 43 kDa=β-Actin.

FIG. 11. EPO protein expression in the culture medium under hypoxicconditions. EPO in the culture medium of Lewis rat kidney cells andHepG2 cells was assessed by western blot. 34 kDa=EPO; 43 kDa=β-Actin.

FIG. 12. Total protein lysates were prepared from rat renal primarycells at passages 1 and 2. Plates from normoxic samples (NC), samples in3% O2 and 7% O2 were processed and run on 10% SDS-PAGE. KNRK cell linewas used as positive control.

FIG. 13. Measuring EPO in media concentrates by western blot. Primarycultured cells from Lewis rats were raised close to confluency at eachpassage on 10 cm plates. The cells were starved with KSFM for 24 hrs andthen placed in a hypoxic chamber (1% O2) for 24, 48 or 72 hrs. Followinghypoxia incubation, the media was collected and concentrated with a 10Kmwco amicon ultra centrifugal device (Millipore). 40 ug of total proteinwas then loaded on a 10% polyacrylamide gel. KNRK cells were used aspositive control.

FIG. 14. Histological analysis of the retrieved implants showed that thekidney cells survived and formed tissue in vivo. Presence of EPOproducing cells were confirmed immunohistochemically using EPO specificantibodies (X400). Left panel: Initial cell density of 1×10⁶cells/injection. Right panel: Initial cell density of 1×10⁶cells/injection. Top row of each panel: 2 weeks. Bottom row of eachpanel: 4 weeks.

FIG. 15. Effect of culture media and hypoxia on renal primary cellsmeasured by real time PCR. Renal primary cells (p0) were grown to 80%confluency in 10 cm plates. Three plates of cells were grown with eitherserum free KSFM or DMEM and placed in a hypoxic chamber at 3% O2. After24 hrs, samples were processed for total RNA and cDNA synthesis. Realtime PCR was done in triplicate, and samples were quantified relative tonormoxic sample.

FIG. 16. Effect of hypoxia on renal primary cells measured by real timePCR. Renal primary cells (passages 0 and 2) were grown to 80% confluencyin 10 cm plates. Cells were then grown in serum free KSFM and placed ina hypoxic chamber at 1% O2. After 24, 48 or 72 hrs, samples wereprocessed for total RNA and cDNA synthesis. Real time PCR was done intriplicate, and samples were quantified relative to normoxic sample.

FIG. 17. Effect of hypoxia on renal primary cells measured by real timePCR. Renal primary cells (passage 0) were grown to 80% confluency in 10cm plates. Cells were then placed in a hypoxic chamber at 1% O2 for upto 24 hrs. Samples were then processed for total RNA and cDNA synthesis.Real time PCR was done in triplicate, and samples were quantifiedrelative to normoxic sample

FIG. 18. Primary human kidney cells were expanded. Shown are cells ofpassages 2, 4, 7 and 9.

FIG. 19. Human primary renal cells were maintained through 20 doublings.

FIG. 20. Human kidney cell characterization. GLEPP1 and EPO positivecells are present in the population.

FIG. 21. Human kidney cell delivery in vivo with a 20 mg/ml collagencarrier. At retrieval, 3 weeks after injection, the injection volume hadbeen maintained, and neovascularization was present.

FIG. 22. Injection of collagen with cultured human kidney cells resultedin EPO expressing tissue formation in vivo.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Cell based therapy for renal failure can be approached in twodirections: total and selective. Described herein is the selective celltherapy approach for achieving restoration of specific functional organcomponents.

The disclosures of all United States patent references cited herein arehereby incorporated by reference to the extent they are consistent withthe disclosure set forth herein. As used herein in the description ofthe invention and the appended claims, the singular forms “a,” “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. Furthermore, the terms “about” and“approximately” as used herein when referring to a measurable value suchas an amount of a compound, dose, time, temperature, and the like, ismeant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% ofthe specified amount. Also, as used herein, “and/or” or “/” refers toand encompasses any and all possible combinations of one or more of theassociated listed items, as well as the lack of combinations wheninterpreted in the alternative (“or”).

“Kidney tissue” is tissue isolated or harvested from the kidney, whichtissue contains kidney cells. In some embodiments, kidney cells arepositive for one or more known kidney markers, e.g., GLEPP1, TammHorsfall, etc. “Cell” or “cells” may be of any suitable species, and insome embodiments are of the same species as the subject into whichtissues produced by the processes herein are implanted. Mammalian cells(including mouse, rat, dog, cat, monkey and human cells) are in someembodiments particularly preferred. “Isolated” as used herein signifiesthat the cells are placed into conditions other than their naturalenvironment. Tissue or cells are “harvested” when initially isolatedfrom a subject, e.g., a primary explant.

“Subjects” are generally human subjects and include, but are not limitedto, “patients.” The subjects may be male or female and may be of anyrace or ethnicity, including, but not limited to, Caucasian,African-American, African, Asian, Hispanic, Indian, etc. The subjectsmay be of any age, including newborn, neonate, infant, child,adolescent, adult, and geriatric.

Subjects may also include animal subjects, particularly mammaliansubjects such as canines, felines, bovines, caprines, equines, ovines,porcines, rodents (e.g., rats and mice), lagomorphs, non-human primates,etc., for, e.g., veterinary medicine and/or pharmaceutical drugdevelopment purposes.

Cells may be syngeneic (i.e., genetically identical or closely related,so as to minimize tissue transplant rejection), allogeneic (i.e., from anon-genetically identical member of the same species) or xenogeneic(i.e., from a member of a different species). Syngeneic cells includethose that are autogeneic (i.e., from the patient to be treated) andisogeneic (i.e., a genetically identical but different subject, e.g.,from an identical twin). Cells may be obtained from, e.g., a donor(either living or cadaveric) or derived from an established cell strainor cell line. Cells may be harvested from a donor, e.g., using standardbiopsy techniques known in the art.

The “primary culture” is the first culture to become established afterseeding disaggregated cells or primary explants into a culture vessel.“Expanding” as used herein refers to an increase in number of viablecells. Expanding may be accomplished by, e.g., “growing” the cellsthrough one or more cell cycles, wherein at least a portion of the cellsdivide to produce additional cells.

“Passaged in vitro” or “passaged” refers to the transfer or subcultureof a cell culture to a second culture vessel, usually implyingmechanical or enzymatic disaggregation, reseeding, and often divisioninto two or more daughter cultures, depending upon the rate ofproliferation. If the population is selected for a particular genotypeor phenotype, the culture becomes a “cell strain” upon subculture, i.e.,the culture is homogeneous and possesses desirable characteristics(e.g., the ability to express EPO).

“Express” or “expression” of EPO means that a gene encoding EPO istranscribed, and preferably, translated. Typically, according to thepresent invention, expression of an EPO coding region will result inproduction of the encoded polypeptide, such that the cell is an “EPOproducing cell.” In some embodiments, cells produce EPO without furthermanipulation such as the introduction of an exogenous gene. In someembodiments, the invention is subject to the proviso that the EPOproducing cells are not manipulated by the introduction of an exogenousgene and/or by an exogenous chemical that stimulates the production ofEPO.

In some embodiments, harvested cells are not passaged. In otherembodiments, cells are passaged once, twice, or three times. In stillother embodiments, cells are passaged more than 3 times. In someembodiments, cells are passaged 0-1, 0-2 or 0-3 times. In someembodiments, cells are passaged 1-2, 1-3, or 1-4 or more times. In someembodiments, cells are passaged 2-3 or 2-4 or more times. In furtherembodiments, cells are passaged 5, 8, 10, 12 or 15 or more times. Insome embodiments, cells are passaged 0, 1, 2, 3 or 4 to 8, 10, 15 or 20or more times. The number of passages used may be selected by, e.g., therelative EPO production measured in the cell population after eachpassage.

Growing and expansion of kidney cells is particularly challengingbecause these cells are prone to the cessation of growth and earlydifferentiation. This challenge is overcome in some embodiments of thepresent invention by using kidney cell specific media that containsadditives that promote their growth. Accordingly, in some embodimentskidney cells are grown in media that includes additives such as growthfactors and other supplements that promote their growth. Further, insome embodiments, EPO producing cells are grown in co-culture with otherrenal cell types.

In some embodiments, kidney cells are grown in Dulbecco's ModifiedEagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) orfetal calf serum (FCS) and, optionally, penicillin-streptomycin (P/S).In other embodiments, kidney cells are grown in keratinocyte serum-freemedium (KSFM). In further embodiments, kidney cells are grown in KSFMwith one or more of the following additives: bovine pituitary extract(BPE) (e.g., 50 g/mL), epidermal growth factor (EGF) (e.g., 5 ng/mL),antibiotic-antimycotic solution (GIBCO) (e.g., 5 mL), fetal bovine serum(FBS) (Gemini Bio-Product) (e.g., 12.5 mL of 2.5%), and insulintransferrin selenium (ITS) (Roche) (e.g., 50 mg for 5 L medium). Asunderstood by those of skill in the art, in some embodiments of theabove media, penicillin-streptomycin (P/S) and antibiotic-antimycoticsolution are interchangeable.

Passaging of kidney cells according to some embodiments may beaccomplished using standard procedures known in the art. For example,the cells may be detached using trypsin/EDTA and transferred to otherplates. This is a standard procedure for many cell types. Briefly, insome embodiments this may be accomplished with the following steps: 1)Remove medium. 2) Add 10 ml PBS/EDTA (0.5 M) for 4 minutes. Confirm theseparation of cell junctions under a phase contrast microscope. 3)Remove PBS/EDTA and add 7 ml Trypsin/EDTA. 4) Add 5 ml medium when80-90% of the cells lift under microscope. 5) Aspirate the cellsuspension into a 15 ml test tube. 6) Centrifuge the cells at 1000 rpmfor 4 minutes. 7) Remove the supernatant. 8) Resuspend cells in 5 ml ofmedium. 9) Pipet out 100 μl of the cell suspension and perform trypanblue stain for viability assay. 10) Count the number of cells onhemocytometer. 11) Aliquot the desired number of cells on the plate andmake the volume of medium to a total of 10 ml. 12) Place the cells inthe incubator.

“Selection” can be based upon any unique properties that distinguish onecell type from another, e.g., density, size, unique markers, uniquemetabolic pathways, nutritional requirements, protein expression,protein excretion, etc. For example, cells may be selected based ondensity and size with the use of centrifugal gradients. Unique markersmay be selected with fluorescent activated cell sorting (FASC),immunomagnetic bead sorting, magnetic activated cell sorting (MASC),panning, etc. Unique metabolic pathways and nutritional requirements maybe exploited by varying the makeup and/or quantity of nutritionalingredients of the medium on which cells are grown, particularly in aserum-free environment. Protein expression and/or excretion may bedetected with various assays, e.g., ELISA.

“EPO producing cell” refers to differentiated cells, of which at least aportion produce EPO (e.g., at least 20, 30, 40, or 50% or more, or morepreferably 60, 70, 80, or 90% or more of the cells produce EPO). In someembodiments, cells produce EPO without further manipulation such as theintroduction of an exogenous gene. In some embodiments, the invention issubject to the proviso that the EPO producing cells are not manipulatedby the introduction of an exogenous gene and/or by an exogenous chemicalthat stimulates the production of EPO. The cells may be harvested from,e.g., the peritubular interstitial cells of the kidney. In someembodiments, the cells are selected for their ability to produce EPO. Inother embodiments, the cells are expanded in number by cell culturetechniques, e.g., passaging. Cells with the specific function of EPOproduction can be used from the kidney and from other sources. Forexample, EPO is also normally produced in the liver.

In the kidney, EPO is generally known to be produced by the interstitialperitubular cells (FIG. 1). In some embodiments, an isolated populationof differentiated kidney cells comprises, consists of or consistsessentially of interstitial peritubular cells of the kidney, consistingof or consisting essentially of 80, 90, 95, or 99 percent or more, ornot more than 20, 10, 5 or 1 percent or less, by number of other celltypes. In other embodiments, the isolated population of differentiatedkidney cells includes other cell types, e.g., endothelial peritubularcells.

In some embodiments, the isolated population of differentiated kidneycells comprises, consists of or consists essentially of kidney cellsthat are selected for EPO production, consisting of or consistingessentially of 80, 90, 95, or 99 percent or more, or not more than 20,10, 5 or 1 percent or less, by number of cells not expressing EPO.Selection may be accomplished by selecting the cells that express EPOusing specific markers. In some embodiments, cells may include varioustypes of kidney cells, so long as the cells express EPO. In furtherembodiments, the entire renal cell colony may be used for expansion andtreatment.

In some embodiments, the isolated population of differentiated kidneycells have a “longevity” such that they are capable of growing throughat least 5, 10, 15, 20, 25 or 30 or more population doublings when grownin vitro. In some embodiments, the cells are capable of proliferatingthrough 40, 50 or 60 population doublings or more when grown in vitro.

“Differentiated” refers to cells or a population containing cells thathave specialized functions, e.g., EPO production and/or expression ofknown markers of differentiated cells (e.g., GLEPP1 and/or Tamm Horsfallkidney cell markers). In this sense they are not progenitor or stemcells. Some embodiments of the present invention are subject to theproviso that harvested differentiated cells are not passaged underconditions to create a population of less specialized cells.

Alternatively, in other embodiments, cells are cultured to produce celllines, which may later be differentiated to produce more specializedcells. The establishment of “cell lines,” as opposed to cell strains,are by and large undifferentiated, though they may be committed to aparticular lineage. Propagation naturally favors the proliferativephenotype, and in some embodiments cells may require a reinduction ofdifferentiation by, e.g., alteration of the culture conditions. Thereare a number of differentiation factors known in the art that may inducedifferentiation in cell lines (e.g., cytokines such as epimorphin andHGF, vitamins, etc.).

Methods of Treatment.

In some embodiments, EPO producing cells are administered to a subjectin need thereof (e.g., by injection) to the kidney (e.g., into thecortex and/or medulla). In other embodiments, EPO producing cells areadministered to other areas of the body, e.g., the liver, peritoneum,etc. In some embodiments, the EPO producing cells are administeredsubcutaneously, subcapsular, etc. In further embodiments, EPO producingcells are administered by implantation of a substrate (e.g., a collagengel scaffold) containing said EPO producing cells described herein. Instill other embodiments, EPO producing cells are administered throughvascular access (e.g., systemically or locally).

Diseases that may be treated with the methods disclosed herein include,but are not limited to, anemias. Anemias include, but are not limitedto, those associated with renal failure or end-stage renal disease,anemias caused by chemotherapies or radiation, anemias of chronicdisorders, e.g., chronic infections, autoimmune diseases, rheumatoidarthritis, AIDS, malignancies, anemia of prematurity, anemia ofhypothyroidism, anemia of malnutrition (e.g., iron deficiency), andanemias associated with blood disorders.

“Treat” refers to any type of treatment that imparts a benefit to apatient, e.g., a patient afflicted with or at risk for developing adisease (e.g., kidney disease, anemia, etc.). Treating includes actionstaken and actions refrained from being taken for the purpose ofimproving the condition of the patient (e.g., the relief of one or moresymptoms), delay in the onset or progression of the disease, etc.

Other endocrine systems may benefit from the therapies disclosed herein,for example, vitamin D producing cell therapy or the angiotensin system.See, e.g., U.S. Patent Application Publication No. 2005/0002915 to Atalaet al., which is incorporated herein by reference. Cells with a specificfunction can be used from the kidney and other sources, i.e., cells thatwould produce target functions. For example, EPO is also normallyproduced in the liver.

Preferably the cells are mixed with or seeded onto a pharmaceuticallyacceptable carrier prior to administration. “Pharmaceuticallyacceptable” means that the compound or composition is suitable foradministration to a subject to achieve the treatments described herein,without unduly deleterious side effects in light of the severity of thedisease and necessity of the treatment. Such formulations can beprepared using techniques well known in the art. See, e.g., U.S. PatentApplication 2003/0180289; Remington: The Science and Practice ofPharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams &Wilkins: Philadelphia, Pa., 2000. The carrier may be a solid or aliquid, or both (e.g., hydrogels), and can be formulated with the cellsas a unit-dose formulation. In some embodiments the cells are providedas a suspension in the carrier to reduce clumping of the cells. In otherembodiments cells are seeded onto a biodegradable scaffold or matrix.

In some embodiments, cells are mixed with a suitable gel foradministration. Suitable gels that may be used in the present inventioninclude, but are not limited to, agars, collagen, fibrin, hydrogels,etc. Besides gels, other support compounds may also be utilized in thepresent invention. Extracellular matrix analogs, for example, may becombined with support gels to optimize or functionalize the gel. One ormore growth factors may also be introduced into the cell suspensions.

Formulations of the invention include those for parenteraladministration (e.g., subcutaneous, intramuscular, intradermal,intravenous, intraarterial, intraperitoneal injection) by injection orimplantation. In one embodiment, administration is carried outintravascularly, either by simple injection, or by injection through acatheter positioned in a suitable blood vessel, such as a renal artery.In some embodiments, administration is carried out by “infusion,”whereby compositions are introduced into the body through a vein (e.g.,the portal vein). In another embodiment, administration is carried outas a graft to an organ or tissue to be augmented as discussed above,e.g., kidney and/or liver.

A “biodegradable scaffold or matrix” is any substance not having toxicor injurious effects on biological function and is capable of beingbroken down into is elemental components by a host. Preferably, thescaffold or matrix is porous to allow for cell deposition both on and inthe pores of the matrix. Such formulations can be prepared by supplyingat least one cell population to a biodegradable scaffold to seed thecell population on and/or into the scaffold. The seeded scaffold maythen implanted in the body of a recipient subject.

In some embodiments, cells are administered by injection of the cells(e.g., in a suitable carrier) directly into the tissue of a subject. Forexample, cells may be injected into the kidney (e.g., the subcapsularspace of the kidney). Because the functional effects of EPO productionwill be systemic, cells may also be administered by injection into othertissues (e.g., the liver, subcutaneously, etc.).

Cells may also be delivered systemically. In further embodiments, cellsare delivered to tissue outside of the kidney (e.g., the liver), as theoutcome of the functional effects of EPO production will be systemic.See, e.g., the “Edmonton protocol,” an established delivery method,where cells are infused into a patient's portal vein (Shapiro et al.(2000) N Engl J Med 343:230-238).

According to some embodiments, the cells administered to the subject maybe syngeneic (i.e., genetically identical or closely related, so as tominimize tissue transplant rejection), allogeneic (i.e., from anon-genetically identical member of the same species) or xenogeneic(i.e., from a member of a different species), as above, with respect tothe subject being treated, depending upon other steps such as thepresence or absence of encapsulation or the administration of immunesuppression therapy of the cells. Syngeneic cells include those that areautogeneic (i.e., from the subject to be treated) and isogeneic (i.e., agenetically identical but different subject, e.g., from an identicaltwin). Cells may be obtained from, e.g., a donor (either living orcadaveric) or derived from an established cell strain or cell line. Asan example of a method that can be used to obtain cells from a donor(e.g., a potential recipient of a bioscaffold graft), standard biopsytechniques known in the art may be employed. Alternatively, cells may beharvested from the subject, expanded/selected in vitro, and reintroducedinto the same subject (i.e., autogeneic).

In some embodiments, cells are administered in a therapeuticallyeffective amount. The therapeutically effective dosage of cells willvary somewhat from subject to subject, and will depend upon factors suchas the age, weight, and condition of the subject and the route ofdelivery. Such dosages can be determined in accordance with proceduresknown to those skilled in the art. In general, in some embodiments, adosage of 1×10⁵, 1×10⁶ or 5×10⁶ up to 1×10⁷, 1×10⁸ or 1×10⁹ cells ormore per subject may be given, administered together at a single time orgiven as several subdivided administrations. In other embodiments, adosage of between 1-100×10⁸ cells per kilogram subject body weight canbe given, administered together at a single time or given as severalsubdivided administration. Of course, follow-up administrations may begiven if necessary.

Cells may be administered according to some embodiments to achieve atarget hematocrit range. The ideal or target hematocrit range may varyfrom subject to subject, depending upon, e.g., specific comorbidities.In some embodiments the target hematocrit is from 30-40%, in someembodiments the target hematocrit is from 33-38%, and in someembodiments the target hematocrit is from 33-36%. Upon administration ofcells according to the present invention, hematocrit may be measuredand, if desired or necessary, corrected by, e.g., further implantationof cells and/or other methods known in the art (e.g., supplementing withrecombinant EPO). Other methods of treatment for anemia and/or renaldisease may be used in conjunction with the methods of treatmentprovided herein, for example, an adapted protein-caloric intake diet.

In further embodiments, if desired or necessary, the subject may beadministered an agent for inhibiting transplant rejection of theadministered cells, such as rapamycin, azathioprine, corticosteroids,cyclosporin and/or FK506, in accordance with known techniques. See,e.g., R. Caine, U.S. Pat. Nos. 5,461,058, 5,403,833 and 5,100,899; seealso U.S. Pat. Nos. 6,455,518, 6,346,243 and 5,321,043. Some embodimentsuse a combination of implantation and immunosuppression, which minimizesgraft rejection. The implantation may be repeated as needed to create anadequate mass of transplanted tissue.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES

Anemia is an inevitable outcome of chronic renal failure due to thekidney's decreased ability to produce erythropoietin (EPO) byperitubular interstitial cells. We investigated whether supplementationof erythropoietin producing cells would be a possible treatment optionfor renal failure-induced anemia by examining the feasibility ofselecting and expanding erythropoietin producing cells for cell-basedtherapy.

The following examples demonstrate that EPO producing cells are presentin renal cells harvested from mouse and rat kidneys. In addition, cellsisolated and expanded using the methods described below include cellsexpressing EPO at every culture stage examined. Further, the actualpercentage of cells expressing the EPO marker in culture was consistentwith the cell population present in normal kidney tissues (seeYamaguchi-Yamada et al., J Vet Med Sci, 67: 891, 2005; Sasaki et al.,Biosci Biotechnol Biochem, 64: 1775, 2000; Krantz, Blood, 77: 419,1991).

Example 1 Expansion of Renal Cell Primary Cultures

Renal cells from 7-10 day old mice C57BL/6 were culture expanded. Mincedkidney (1 kidney of mouse) was placed into a 50 cc tube with 15 ml ofcollagenase/dispase (0.2 mg/ml). The kidney tissue fragments wereincubated in a 37° C. shaker for 30 min with collagenase/dispase mix(0.2 mg/ml; 15 ml). Sterile PBS with Gelatin (20 ml), was added (withGelatin (DIFCO) 2 mg/ml) to the digestion solution. The mixture wasfiltered thorough a 70 micron filter to remove undigested tissuefragments. The collected solution was mixed well (being careful not tomake air bubbles), and divided into two 50 cc tubes. The tubes werecentrifuged at 1000(−1500) RPM for 5 min. The supernatant was discardedand the pellet of each tube was resuspended in 3 ml of KSFM medium. DMEMmedium (10% FBS, 5 ml P/S) is used for stromal cells, and KSFM with BPE,EGF, 5 ml antibiotic-antimycotic, 12.5 ml FBS (Gemini Bio-Product,2.5%), Insulin Transferrin Selenium (Roche) (50 mg for 5 L medium) withBPE and EGF for epithelial components. P/S or antibiotic-antimycotic(GIBCO) may also be added. Each tissue was seeded on to a 25 mm plateand medium was added (total 3 ml).

Cells were maintained by changing the medium the next day, and thenevery 2 days depending on the cell density. Cells were passaged whenthey were 80-90% confluent by detachment using trypsin/EDTA andtransferred to other plates with the following steps: 1) Remove medium.2) Add 10 ml PBS/EDTA (0.5 M) for 4 minutes. Confirm the separation ofcell junctions under a phase contrast microscope. 3) Remove PBS/EDTA andadd 7 ml Trypsin/EDTA. 4) Add 5 ml medium when 80-90% of the cells liftunder microscope. 5) Aspirate the cell suspension into a 15 ml testtube. 6) Centrifuge the cells at 1000 rpm for 4 minutes. 7) Remove thesupernatant. 8) Resuspend cells in 5 ml of medium. 9) Pipet out 100 μlof the cell suspension and perform trypan blue stain for viabilityassay. 10) Count the number of cells on hemocytometer. 11) Aliquot thedesired number of cells on the plate and make the volume of medium to atotal of 10 ml. 12) Place the cells in the incubator.

Alternatively, the following protocol was used. Kidneys from 10 day oldmale C57BL/6 mice were collected in Krebs buffer solution (SigmaAldrich, St. Louis, Mo. USA) containing 10% antibiotic/antimycotic(Gibco Invitrogen, Carlsbad, Calif. USA) to avoid risk of contamination.The kidneys were immediately transported to a culture hood where thecapsule was removed. The medullary region of the kidney was removed, andonly the cortical tissue was used to isolate cells that had beenpreviously identified as EPO producing cells (Maxwell et al., KidneyInternational, 44: 1149, 1993). The kidney tissue was minced andenzymatically digested using Liberase Blendzyme (Roche, Mannheim,Germany) for 25 minutes at 37 degrees Celsius in a shaking water bath.The supernatant was removed and the cell pellet was passed through a 100μm cell strainer to obtain a single cell suspension for culture.

Subsequently, the cells were plated at a density of 5×10⁵ cells/ml in 10cm tissue culture treated plates filled with culture media. The culturemedia consisted of a mixture of keratinocyte serum-free medium (KSFM)and premixed Dulbecco's Modified Eagle's Medium (DMEM) at a ratio of1:1. The premixed DMEM media contained ¾ DMEM and ¼ HAM's F12 nutrientmixture supplemented with 10% fetal bovine serum (FBS), 1%Penicillin/Streptomycin, 1% glutamine 100× (Gibco), 1 ml of 0.4 μg/mlhydrocortisone, 0.5 ml of a 10⁻¹⁰ M cholera toxin solution, 0.5 ml of a5 mg/ml insulin solution, 12.5 ml/500 ml of a 1.2 mg/ml adeninesolution, 0.5 ml of a 2.5 mg/ml transferrin+0.136 mg/ml triiodothyroninemixture, and 0.5 ml of a 10 μg/ml epidermal growth factor (EGF)solution. All tissue culture reagents were purchased from Sigma-Aldrich(St. Louis, Mo. USA) unless otherwise stated. The cells were incubatedat 37° C. under 5% CO₂ with medium change every 3 days, and the cellswere subcultured for expansion at a ratio of 1:3 when confluent.

Example 2 Characterization for EPO Production

The cells from early passages (1, 2 and 3) were characterized for EPOexpression using immunocytochemistry and western blot analysis withspecific antibodies (rabbit polyclonal anti-EPO antibodies, sc-7956,Santa Cruz Technologies, Santa Cruz, Calif.).

Renal cells were plated in 8-well chamber slides at a density of 3000cells per well. The cells were incubated at 37° C. under 5% CO₂ for 24 hto allow attachment. This was followed by fixation with 4%paraformaldehyde for 10 minutes at room temperature. Permeabilization ofcell membranes was performed by adding 0.1% Triton-X 100 in PBS for 3minutes at room temperature. Cells were then incubated in goat serum for30 minutes at room temperature. After washing, cells were incubated withthe primary antibodies for 1 h (1:50) at room temperature. Cells werewashed a second time and biotinylated goat polyclonal anti-rabbitantibodies (polyclonal anti rabbit IgG, Vector Laboratories, Inc.,Burlingame, Calif.) (1:200) were added, followed by incubation at roomtemperature for 45 minutes. Chromogenic detection of EPO followed afinal washing step and was performed using the Vector ABC kit accordingto the manufacturer's instructions (Vector Laboratories, Inc.,Burlingame, Calif.). Slides without the primary antibodies served asinternal negative controls, and normal mouse renal tissue served as thepositive control.

Renal cells in culture showed multiple phenotypes under the microscope.The cells reached confluency within 7 to 10 days of plating. Many of thecells observed in the first 3 passages after isolation from the kidneystained positively for EPO, as compared to the negative controls, whichshowed no background or nonspecific staining (FIG. 2), which indicatedthat the observed staining was likely due to the presence of EPO in thecultures. The number of cells that stained positively for EPO remainedconstant throughout the 3 passages studied, even when phenotypic changeswere observed in the culture during the same time period.Immunohistochemical staining of kidney tissue indicated a similar amountof EPO expression as that found in cultured cells (FIG. 3).

The number of cells expressing EPO decreased slightly with subsequentpassages (FIG. 4). This is most likely due to the increased number ofpassages and loss of cells/function over time and manipulation. However,the relative percentage appears to remain stable after the firstpassage.

EPO expression was also confirmed by western blot, shown in FIG. 5.

Example 3 Mouse and Rat Renal Cell Characterization

FACS analysis was used to quantify the number of EPO-producing cells inthe established renal cell cultures at each passage (1-3 passages). Thecells were collected by trypsinization and centrifugation, resuspendedin media, and passed through a 70 μm cell strainer to ensure a singlecell suspension. After counting the cells, they were spun down andresuspended in PBS at 5-7.5×10⁵ cells/tube to remove FBS from the cells.The cells were fixed with 2% formaldehyde for 10 minutes at 4° C. andpermeabilized using 100% methanol for 10 minutes at room temperature.Subsequently, the cells were resuspended in 3% goat serum in PBSfollowed by incubation with the rabbit anti-EPO primary antibody sc-7956(Santa Cruz Biotechnology, Santa Cruz, Calif.) for 45 minutes on ice.Cells were washed twice with 3% goat serum in PBS prior to incubationwith fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbitsecondary antibodies for 1 hour. The cells were then washed thoroughlywith 3% serum in PBS and transferred to the FACS machine (FACS CaliburE6204, Becton-Dickinson, Franklin Lakes, N.J.).

Fluorescent activated cell sorting experiments demonstrated that 44% ofpassage 1 (P1) cells were EPO positive. This percentage increased to 82%at passage 2 (P2), and then dropped back to 42% at passage 3 (P3). Thismay indicate that, during the first few days of culture, proliferationof EPO-producing cells and/or upregulation of EPO gene expression occursin response to the lower oxygen concentration in the media compared tonormal living tissue. These responses could then normalize over the nextfew days, resulting in numbers of EPO-producing cells that are close tothose found in renal tissue (FIG. 6, top row).

The FACS data demonstrate the maintenance of EPO expression over severalpassages. It should be noted that there was a surge in the number ofcells expressing EPO (82%) in the passage 2 culture, which was confirmedby several repeat experiments. Though not wishing to be bound to anyparticular theory, one possible explanation for this phenomenon could bethat EPO expression is an inherent trait of all renal cells that can beturned on and off as needed. In this case, following the abrupt changein survival conditions between the body and the culture plate, the cellsmay have been driven to express EPO momentarily until stabilization ofthe culture occurred. Consistent with this, the EPO surge was quicklyreversed and passage 3 analyses showed a lower percentage of EPOproducing cells (42%).

Mouse cell characterization by immunofluorescence confirmed EPOexpression (FIG. 7A). The population of cells was positive for thekidney cell markers GLEPP1 and Tamm Horsfall (FIG. 7B).

Rat cell passages 0, 1 and 2 were also analyzed for EPO production usingfluorescence activated cell sorting (FACS) (FIG. 6, bottom row).Cultured rat cells had various cell morphologies and were positive forGLEPP1 and Tamm Horsfall kidney cell markers (FIG. 8).

Example 4 Exposure of EPO Producing Cultures to Hypoxic Conditions

While maintenance of phenotypic characteristics is essential during cellexpansion stages, a critical component that ensures the success of celltherapy is the ability of EPO producing cells to regulate and maintainnormal EPO levels. EPO belongs to the hematopoietic cytokine family, andit controls erythropoiesis in bone marrow, and regulates theproliferation, differentiation and survival of erythroid progenitorcells through EPO receptor (EPOR)-mediated signal transduction. EPO islargely produced in the kidney, and when this organ fails, EPOproduction falls, leading to anemia. EPO expression in the body dependslargely on the oxygen tension in the environment surrounding the cellscapable of producing EPO. Factors influencing oxygen levels include lackof oxygen in the ambient air and decreased renal blood flow.

To determine whether the EPO expressing cells in culture could respondto changing oxygen levels, an experiment was performed in which thecells were serum-starved for 24 hours followed by exposing them tovarious levels of oxygen in vitro. Lewis rat kidney cells and HepG2(human hepatocellular liver carcinoma cell line) cells were culturedunder normal and hypoxic conditions, and EPO production was assessed andconfirmed by western blot of cells. EPO presence in the culture mediumwas also measured and confirmed by analyzing the supernatants fromcultured renal cells under normoxic and hypoxic conditions with thedouble antibody sandwich enzyme-linked immunosorberbent assay using aQuantikine® IVD® Erythropoietin ELISA kit (R&D Systems®, Minneapolis,Minn.).

The cells were placed in serum free media for 24 hours prior to theexperiment. The plates were then transferred to a hypoxic chamber andexposed to different hypoxic conditions (1%, 3%, 5%, and 7% oxygen).HepG2 cells were used as positive controls, as they have been previouslyreported to produce high levels of EPO in culture (Horiguchi et al.,Blood, 96: 3743). EPO expression by HepG2 was confirmed by western blot(FIG. 9). All cells were harvested in lysis buffer containing NP-40.Protein concentration in each sample was measured using a Bio-Radprotein assay. 40 μg total protein was run out on a 10% acrylamide gelusing SDS-PAGE. Proteins were then transferred onto a PVDF membrane(Millipore Corp.). Detection of β-actin expression in the lysates wasused as the loading control. EPO antibody (rabbit polyclonal sc-7956,Santa Cruz Biotechnology) was used at 1:200 and the secondary antibody(goat anti-rabbit 7074, Cell Signaling Technology, Beverly, Mass.) wasused at 1:2000. To measure the amount of EPO secreted into the media bythe primary renal cultures, the media was collected and concentrateddown to 500 ul using an Amicon Ultra centrifugal filter device(Millipore Corporation, Billerica, Mass.). Samples of this media wererun on a 10% polyacrylamide gel. EPO antibody (rabbit polyclonalsc-7956, Santa Cruz Biotechnology) was used at 1:100 and the secondaryantibody (goat anti-rabbit 7074, Cell Signaling Technology, Beverly,Mass., USA) was used at 1:2000.

Western blotting showed a slight increase in the EPO expression in thecell lysate after hypoxia (FIG. 10). These results, however, were notseen when media concentrates were used to measure EPO (FIG. 11). Themedia testing indicated that all media concentrates (hypoxic andnormoxic conditions) contained the same low amount of EPO.

Alternatively, total protein lysates were prepared from rat renalprimary cells at passages 1 and 2. Plates from normoxic samples (NC),samples in 3% O2 and 7% O2 were processed and Run on 10% SDS-PAGE. TheKNRK cell line was used as positive control. Results are shown in FIG.12.

Without wishing to be bound by any particular theory, this may indicatethat 24 hours might not be enough time for secreted EPO levels to riseto a level that is detectable by western blot. It is likely that alonger exposure time would be required for the cells to begin to secreteEPO, as de novo protein production may take several hours to becomeapparent. Therefore the following experiment was performed, in whichcells were placed in hypoxic conditions for 24, 48 and 72 hours.

Primary cultured cells from Lewis rats were raised close to confluencyat each passage on 10 cm plates. The cells were placed in a hypoxicchamber (1% O₂) for 24, 48 or 72 hrs. Following hypoxia incubation, themedia was collected and concentrated with a 10K molecular weight cutoffAmicon Ultra centrifugal device (Millipore). 40 μg of total protein wasthen loaded on a 10% Polyacrylamide gel. KNRK cells were used as apositive control. Results are shown in FIG. 13.

In summary, all experiments indicated that the EPO levels in primaryculture cells were greater than or equal to those measured in the HepG2positive controls, and the EPO producing cells are able to respond tochanging environment.

Example 5 Administration of EPO Producing Cells in Vivo

To determine whether EPO producing cells survive and form the tissues invivo, renal cells mixed in collagen gel were implanted subcutaneously inathymic mice at concentrations of 1×10⁶ and 5×10⁶ followed by retrievalat 14 and 28 days after implantation for analysis. Cells at differentpassages from 1-5 were used. The cells were suspended in a collagen gelfor easy injection (concentration: 0.1 mg/ml).

Histologically, the retrieved implants showed that surviving renal cellscontinue expressing EPO proteins, confirmed immunohistochemically usingEPO specific antibodies (FIG. 14).

These results demonstrate that EPO producing renal cells grown andexpanded in culture stably expressed EPO in vivo. Thus, EPO producingcells may be used as a treatment option for anemia caused by chronicrenal failure.

Example 6 Analysis of EPO Expression with Real Time PCR

Real time PCR was performed to assess rat cell expression of EPO inresponse to hypoxic conditions.

To test the effect of culture media, cells grown in either KSFM and DMEMwere exposed to hypoxic conditions (3% O₂). Renal primary cells (passage0) were grown to 80% confluency in 10 cm plates. Three plates of cellswere grown with either serum free KSFM or DMEM and placed in a hypoxicchamber at 3% O₂. After 24 hrs, samples were processed for total RNA andcDNA synthesis. Real time PCR was done in triplicate, and samples werequantified relative to normoxic sample. Results are shown in FIG. 15.

Rat kidney culture EPO expression was compared with real time PCR across24, 48 and 72 hours. Renal primary cells (passages 0 and 2) were grownto 80% confluency in 10 cm plates. Cells were then grown in serum freeKSFM and placed in a hypoxic chamber at 1% O2. After 24, 48 or 72 hours,samples were processed for total RNA and cDNA synthesis. Real time PCRwas done in triplicate, and samples were quantified relative to normoxicsample. Results are shown in FIG. 16.

Testing timepoints for up to 24 hours, renal primary cells (passage 0)were grown to 80% confluency in 10 cm plates. Cells were then placed ina hypoxic chamber at 1% O2 for up to 24 hours. Samples were thenprocessed for total RNA and cDNA synthesis. Real time PCR was run intriplicate, and samples were quantified relative to normoxic sample.Results are shown in FIG. 17.

Example 7 Expansion of Human Kidney Cells

The growth and expandability of primary human kidney cells were alsodemonstrated using the media and conditions described above. Culturesfrom passages 2, 4, 7 and 9 are shown in FIG. 8. It was demonstratedthat human primary renal cells can be maintained through twentydoublings (FIG. 19). Human kidney cell cultures were characterized forEPO and GLEPP1 expression (FIG. 20).

Example 8 Human Kidney Cell Delivery Via Collagen Injection

Human renal cells mixed in collagen gel were implanted subcutaneously inathymic mice as described above in Example 5. Collagen concentrations of1 mg/ml, 2 mg/ml and 20 mg/ml were compared. At 1 and 2 mg/ml, the invivo volume disappeared after administration. At 20 mg/ml, the in vivoinjection volume was maintained, and neo-vascularization was seen FIG.21. Histology confirmed that EPO expressing tissue was formed in vivo(FIG. 22).

Example 9 EPO Producing Cell Selection with Magnetic Cell Sorting

Cells are selected for EPO production using magnetic cell sorting. Asingle-cell suspension is isolated using a standard preparation method.After preparation of single-cell suspension, count the total number ofthe cells and centrifuge cell samples to obtain a pellet. Block thecells with 10% of goat serum (of animal where the secondary antibody ismade) for 10 minutes. Add 1 or 2 mL of the blocking solution. After 10minutes of centrifugation, resuspend the cells in the primary antibodyfor EPO (use 1 μg of the primary antibody/million of cells). Typically,label for 15 minutes at 4-8° C. is sufficient. Wash the cells twice toremove any unbound primary antibody with 1-2 mL of buffer per 10⁷ cellsand centrifuge at 300×g for 10 minutes. After two successive washes, thepellet is resuspended in 80 μL of PBS (0.5% of BSA and 2 mM of EDTA, pH7.2) per 10⁷ cells. Add 20 μL of Goat Anti-Rabbit MicroBeads per 10⁷cells. Mix well and incubate for 15 minutes at 4-8° C. Wash the cellstwice by adding 1-2 mL of buffer per 10⁷ cells and centrifuge at 300×gfor 10 minutes. Pipette off supernatant completely. Resuspend up to 10⁸cells in 500 μL of buffer (Note: For higher cell numbers, scale upbuffer volume accordingly; for depletion with LD Columns, resuspend cellpellet in 500 μL of buffer for up to 1.25×108 cells). Proceed tomagnetic cell separation

Note: Work fast, keep cells cold, and use pre-cooled solutions. Thiswill prevent capping of antibodies on the cell surface and non-specificcell labeling. Volumes for magnetic labeling given below are for up to10⁷ total cells. When working with fewer than 10⁷ cells, use the samevolumes as indicated. When working with higher cell numbers, scale upall reagent volumes and total volumes accordingly (e.g. for 2×10⁷ totalcells, use twice the volume of all indicated reagent volumes and totalvolumes). Working on ice may require increased incubation times. Highertemperatures and/or longer incubation times lead to non-specific celllabeling.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of producing an isolatedpopulation of erythropoietin (EPO) producing cells, said methodcomprising the steps of: providing differentiated mammalian kidney cellscomprising peritubular interstitial cells; growing said peritubularinterstitial cells in vitro in co-culture with multiple other kidneycell types; passaging said differentiated mammalian kidney cells invitro from 2 to 9 times, wherein said peritubular interstitial cellsproduce EPO after said passaging; and then, selecting saiddifferentiated mammalian kidney cells for a subpopulation of cellscomprising said peritubular interstitial cells producing EPO, whereinsaid selecting comprises selection based upon density and size, therebyproducing an isolated population of EPO producing cells, subject to theproviso that the cells of said population of EPO producing cells are nottransfected with an exogenous DNA encoding a polypeptide.
 2. The methodof claim 1, wherein said passaging step comprises growth of saiddifferentiated mammalian kidney cells in a medium comprising insulintransferrin selenium (ITS).
 3. The method of claim 1, wherein saiddifferentiated mammalian kidney cells of said providing step furthercomprises endothelial cells of the kidney.
 4. The method of claim 1,wherein said selecting comprises the use of centrifugal gradients. 5.The method of claim 1, wherein said passaging is carried out from 2 to 5times.
 6. The method of claim 1, wherein said passaging is carried outfrom 3 to 5 times.
 7. The method of claim 1, wherein said populationproduces EPO without manipulation with an exogenous chemical orexogenous gene that stimulates production of EPO.
 8. The method of claim1, wherein said population produces EPO under normoxic conditions invitro without manipulation with an exogenous chemical or exogenous genethat stimulates production of EPO.
 9. The method of claim 1, whereinsaid cells are human.
 10. The method of claim 1, wherein said growing iscarried out under hypoxic conditions.
 11. The method of claim 1, whereinsaid differentiated mammalian kidney cells are positive for GLEPP1and/or Tamm Horsfall after said passaging.
 12. The method of claim 1,said method further comprising providing the isolated population of EPOproducing cells in a pharmaceutically acceptable carrier.
 13. The methodof claim 12, wherein said pharmaceutically acceptable carrier issuitable for injecting or implanting into a patient.
 14. The method ofclaim 12, wherein said pharmaceutically acceptable carrier comprises abiodegradable scaffold suitable for implanting into a kidney of apatient.
 15. The method of claim 12, wherein said pharmaceuticallyacceptable carrier is a gel.
 16. The method of claim 15, wherein the gelis an agar gel, a collagen gel, a fibrin gel, or a hydrogel.
 17. Themethod of claim 13, wherein said cells are autogeneic with respect tothe patient.
 18. The method of claim 14, wherein said cells areautogeneic with respect to the patient.